Detection of Interleukin-1 ß (IL-1ß) in Single Human Blastocyst-Conditioned Medium Using Ultrasensitive Bead-Based Digital Microfluidic Chip and Its Relationship with Embryonic Implantation Potential.

The implantation of human embryos is a complex process involving various cytokines and receptors expressed by both endometrium and embryos. However, the role of cytokines produced by a single embryo in successful implantation is largely unknown. This study aimed to investigate the role of IL-1ß expressed in a single-embryo-conditioned medium (ECM) in embryo implantation. Seventy samples of single ECM were analyzed by a specially designed magnetic-beads-based microfluidic chip from 15 women. We discovered that IL-1ß level increased as the embryo developed, and the difference was significant. In addition, receiver operator characteristic (ROC) curves analysis showed a higher chance of pregnancy when the IL-1ß level on day 5 ECM was below 79.37 pg/mL and the difference between day 5 and day 3 was below 24.90 pg/mL. Our study discovered a possible association between embryonic proteomic expression and successful implantation, which might facilitate single-embryo transfer in the future by helping clinicians identify the embryo with the greatest implantation potential.

Programmable UV-Curable Resin by Dielectric Force.

In this study, UV-curable resin was formed into different patterns through the programmable control of dielectric force. The dielectric force is mainly generated by the dielectric chip formed by the interdigitated electrodes. This study observed that of the control factors affecting the size of the UV resin driving area, current played an important role. We maintained the same voltage-controlled condition, changing the current from 0.1 A to 0.5 A as 0.1 A intervals. The area of droplets was significantly different at each current condition. On the other hand, we maintained the same current condition, and changed the voltage from 100 V to 300 V at 50 V intervals. The area of droplets for each voltage condition was not obviously different. The applied frequency of the AC (Alternating Current) electric field increased from 10 kHz to 50 kHz. After driving the UV resin, the pattern line width of the UV resin could be finely controlled from 224 um to 137 um. In order to form a specific pattern, controlling the current and frequency could achieved a more accurate shape. In this article, UV resin with different patterns was formed through the action of this dielectric force, and after UV curing, tiny structural parts could be successfully demonstrated.

Terahertz Combined with Metamaterial Microfluidic Chip for Troponin Antigen Detection.

In this paper, we use terahertz combined with metamaterial technology as a powerful tool to identify analytes at different concentrations. Combined with the microfluidic chip, the experimental measurement can be performed with a small amount of analyte. In detecting the troponin antigen, surface modification is carried out by biochemical binding. Through the observation of fluorescent antibodies, the average number of fluorescent dots per unit of cruciform metamaterial is 25.60, and then, by adjusting the binding temperature and soaking time, the average number of fluorescent dots per unit of cruciform metamaterial can be increased to 181.02. Through the observation of fluorescent antibodies, it is confirmed that the antibodies can be successfully stabilized on the metamaterial and then bound to the target antigen. The minimum detectable concentration is between 0.05~0.1 µg/100 µL, and the concentration and ΔY show a positive correlation of R2 = 0.9909.

Contactless Micro-Droplet Manipulation of Liquid Released from a Parallel Plate to an Open Region in Electrowetting-on-Dielectric Platform.

In electrowetting-on-dielectric (EWOD) platform, the transfer of droplets from the EWOD boundary region (top plate and bottom plate) to the open region is challenging. The challenge is due to the resistance-like surface tension, friction from the top-plate edge, and the so-called boundary. For this purpose, we designed the top plate to minimize the friction resistance at the boundary. The experiment focused on Gibb's formula and successfully transferred the liquid droplet between the top plate and bottom plate boundary region under a high voltage environment. The threshold voltage for the successful transportation of the droplet between the boundary is 250 V which provides strong pressure to drive the droplet.

Sensitivity Enhancement and Probiotic Detection of Microfluidic Chips Based on Terahertz Radiation Combined with Metamaterial Technology.

Terahertz (THz) radiation has attracted wide attention in recent years due to its non-destructive properties and ability to sense molecular structures. In applications combining terahertz radiation with metamaterial technology, the interaction between the terahertz radiation and the metamaterials causes resonance reactions; different analytes have different resonance performances in the frequency domain. In addition, a microfluidic system is able to provide low volume reagents for detection, reduce noise from the environment, and concentrate the sample on the detection area. Through simulation, a cruciform metamaterial pattern was designed; the proportion, periodicity, and width of the metamaterial were adjusted to improve the sensing capability of the chip. In the experiments, the sensing capabilities of Type A, B, and C chips were compared. The Type C chip had the most significant resonant effect; its maximum shift could be increased to 89 GHz. In the probiotic experiment, the cruciform chip could have a 0.72 GHz shift at a concentration of 0.025 mg/50 µL, confirming that terahertz radiation combined with a metamaterial microfluidic chip can perform low-concentration detection.

DNA Sequencing from Subcritical Concentration of Cell-Free DNA Extracted from Electrowetting-on-Dielectric Platform.

Electro-Wetting-On-Dielectric (EWOD) based digital operations have demonstrated outstanding potential in actuating and manipulating liquid droplets. Here, we adapted the EWOD for extracting femtogram quantities of cell-free DNA (cf-DNA) from 1 µL of KSOM mouse embryo culture medium. Our group extracted the femtogram quantity of cf-DNA from 1 µL of mouse embryo culture medium in our previous work. Here, we initially explain a modification from our previous extraction protocol, which improves the extraction percentage to 36.74%. Though the modified extraction protocol improves the extraction percentage from our previously reported work, the quantity is still in the femtogram range. The cf-DNA in femtogram quantity is in subcritical/subthreshold concentration for any further analysis, such as sequencing. To the best of our knowledge, we need a minimum of picogram/nanogram DNA quantities for further analysis. We demonstrated a ground-breaking mechanism of this subcritical concentration of cf-DNA amplification to the nanogram range and performed DNA sequencing. Basic Local Alignment Search Tool (BLAST) is used as a sequence similarity search program to confirm the identity percentage between query and subject. More than 97% of nucleotide identities between query and subject sequences have been obtained from the sequencing result. Hence, we can use the methodology to amplify the subcritical concentration of extracted DNA for further analytics. Moreover, as we extract the cf-DNA from the embryo culture medium, the natural growth of the embryo has not been disrupted. This entire mechanism will pave a new path towards the lab-on-a-chip (LOC) concept.

Microfluidic Microalgae System: A Review.

Microalgae that have recently captivated interest worldwide are a great source of renewable, sustainable and economical biofuels. The extensive potential application in the renewable energy, biopharmaceutical and nutraceutical industries have made them necessary resources for green energy. Microalgae can substitute liquid fossil fuels based on cost, renewability and environmental concern. Microfluidic-based systems outperform their competitors by executing many functions, such as sorting and analysing small volumes of samples (nanolitre to picolitre) with better sensitivities. In this review, we consider the developing uses of microfluidic technology on microalgal processes such as cell sorting, cultivation, harvesting and applications in biofuels and biosensing.

Identification of Microorganisms Using an EWOD System.

Among the advantages of an electrowetting-on-dielectric (EWOD) chip are its uncomplicated fabrication and low cost; one of its greatest strengths that might be applied in the field of biomedical technology is that it can accurately control volume and reduces the amount of samples and reagents. We present an EWOD for the biochemical identification of microorganisms, which is required to confirm the source of microbial contamination or quality inspection of product-added bacteria, etc. The traditional kit we used existed in the market; the detection results are judged by the pattern of color change after incubation. After a preliminary study, we confirmed that an image-processing tool (ImageJ) provides a suitable method of analysis, and that, when the concentration of the sugar reagent is 38 µg/µL, the best operating parameters for the EWOD chip in silicone oil are 40 V and 1.5 kHz. Additionally, we completed the biochemical identification of five bacterial species on the EWOD chip at the required concentration of the kit. Next, we found a decreased duration of reaction and that the least number of bacteria that were identifiable on the chip lies between 100 and 1000 CFU per droplet. Because the number of bacteria required on the chip is much smaller than for the kit, we tested whether a single colony can be used for identification, which provided a positive result. Finally, we designed an experimental flow to simulate an actual sample in an unclean environment, in which we divided the various processed samples into four groups to conduct experiments on the chip.

Detection of the Freshness of Kiwifruit With a TD-GC-MS and a Gas-Sensing Array Based on the Surface-Acoustic-Wave Technique.

An electronic nose is an arrayed gas sensor mimicking the human olfactory system that can analyze and identify a flavour on collecting an odour from an environment. In our experiments, an electronic-nose system based on a surface acoustic wave (SAW) was used to measure the freshness of kiwifruit. 128° YX-LiNbO3 acted as a piezoelectric material; Au was deposited as an electrode and sensing area. With a polymer coating of various types on the sensing area and a connection to an oscillator circuit, a 113-114 MHz SAW was obtained. Depending on the properties of varied polymers, the frequency shift varied due to absorbed volatile organic compounds (VOC). In this way, with four surface-acoustic-wave sensors coated with varied polymers we built a kiwi-flavour database according to results from a TD-GC-MS system. When the concentration of esters increased, the kiwifruit began to ripen, accompanied by increased concentrations and types of VOC. As a result, polystyrene (PS) and fluoropolymer (CYTOP) polymers, which played the role of sensing materials, served as major materials to determine the ester aroma profile. Polyvinyl alcohol (PVA), polyvinyl butyral (PVB) and poly-N-vinylpyrrolidone (PNVP) were used to trap the alcohols and acids during a kiwifruit ripening period. This research proved that discrimination of differences is feasible from an unripe stage to a ripe stage and from a ripe stage to an over-ripe stage.

A High-Voltage TENG-Based Droplet Energy Generator With Ultralow Liquid Consumption.

A solid-liquid triboelectric nanogenerator (TENG) has attracted increasing research interest in relation to the development of regeneration energy based on water resources. The output of solid-liquid TENG remains unsolved, however, because of the low voltage output that impedes wide applications. To this end, in this work we developed a miniaturized microfluidic channel-based TENG device for highly efficient conversion of energy from the transport of a water droplet to an electrical output. We investigated an optimized design in a triboelectric material, the droplet transport and the electrostatic induction layer to provide a high voltage output and stable energy harvesting. The optimized device demonstrated maximum voltage amplitude 102 mV with an ultralow liquid consumption, 0.36 [Formula: see text], resulting in sample-energy conversion 283.33 mV/ [Formula: see text]. This novel device is expected potentially to address the limitations imposed by sample consumption in energy harvesting in the future.

Effects of Electromagnets on Bovine Corneal Endothelial Cells Treated with Dendrimer Functionalized Magnetic Nanoparticles.

To improve bovine corneal endothelial cell (BCEC) migration, enhance cell energy, and facilitate symmetric cell distribution in corneal surfaces, an electromagnet device was fabricated. Twenty nanometer superparamagnetic iron oxide nanoparticles (SPIONs) functionalized with fourth-generation dendrimer macromolecules were synthesized, and their size and structure were evaluated using transmission electron microscopy (TEM), X-ray photoelectron spectroscopy (XPS), and Fourier transform infrared spectroscopy (FTIR). The results confirmed the configuration of the dendrimer on the SPION surfaces. In vitro biocompatibility was assessed using the 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyl tetrazolium bromide assay. No significant toxicity was noted on BCECs within 24 h of incubation. In the cell migration assay, cells treated with dendrimer-coated SPIONs exhibited a relatively high wound healing rate under sample addition (1 µg/mL) under a magnetic field. Real-time PCR on BCECs treated with dendrimer-coated SPIONs revealed upregulation of specific genes, including AT1P1 and NCAM1, for BCECs-dendrimer-coated SPIONs under a magnetic field. The three-dimensional dispersion of BCECs containing dendrimer-coated SPIONs under a magnetic field was evaluated using COMSOL Multiphysics software. The results revealed the BCECs-SPION vortex pattern layers in the corneal surface corresponded to the electromagnet's displacement from the ocular surface. Magnetic resonance imaging (MRI) indicated that dendrimer-coated SPIONs can be used as a T2 contrast agent.

Virtual Stencil for Patterning and Modeling in a Quantitative Volume Using EWOD and DEP Devices for Microfluidics.

Applying microfluidic patterning, droplets were precisely generated on an electrowetting-on-dielectric (EWOD) chip considering these parameters: number of generating electrodes, number of cutting electrodes, voltage, frequency and gap between upper and lower plates of the electrode array on the EWOD chip. In a subsequent patterning experiment, an environment with three generating electrodes, one cutting electrode and a gap height 10 µm, we obtained a quantitative volume for patterning. Propylene carbonate liquid and a mixed colloid of polyphthalate carbonate (PPC) and photosensitive polymer material were manipulated into varied patterns. With support from a Z-axis lifting platform and a UV lamp, a cured 3D structure was stacked. Using an EWOD system, a multi-layer three-dimensional structure was produced for the patterning. A two-plate EWOD system patterned propylene carbonate in a quantitative volume at 140 Vpp/20 kHz with automatic patterning.

A Review on Microfluidics: An Aid to Assisted Reproductive Technology.

Infertility is a state of the male or female reproductive system that is defined as the failure to achieve pregnancy even after 12 or more months of regular unprotected sexual intercourse. Assisted reproductive technology (ART) plays a crucial role in addressing infertility. Various ART are now available for infertile couples. Fertilization in vitro (IVF), intracytoplasmic sperm injection (ICSI) and intrauterine insemination (IUI) are the most common techniques in this regard. Various microfluidic technologies can incorporate various ART procedures such as embryo and gamete (sperm and oocyte) analysis, sorting, manipulation, culture and monitoring. Hence, this review intends to summarize the current knowledge about the application of this approach towards cell biology to enhance ART.

Extracellular and intracellular intermittent magnetic-fluid hyperthermia treatment of SK-Hep1 hepatocellular carcinoma cells based on magnetic nanoparticles coated with polystyrene sulfonic acid.

The use of magnetic nanoparticles (MNPs) magnetized on applying an alternating magnetic field (AMF) to stimulate the thermal characteristics and to induce tumor apoptosis is a currently active area of research in cancer treatment. In previous work, we developed biocompatible and superparamagnetic polystyrene-sulfonic-acid-coated magnetic nanoparticles (PSS-MNPs) as applications for magnetically labeled cell trapping, but without assessment of treatment effects on tumor diseases. In the present work, we examined PSS-MNP-induced magnetic fluid hyperthermia (MFH) on SK-Hep1 hepatocellular carcinoma (HCC) cells for lethal thermal effects with a self-made AMF system; an adjustable AMF frequency generated a variable intensity of magnetic field and induced MNP relaxation. The extracellular and intracellular MFH treatments on a SK-Hep1 cell line were implemented in vitro; the result indicates that the lethal effects were efficient and caused a significantly decreased cell viability of SK-Hep1 cells. As the PSS-MNP concentration decreased, especially in intracellular MFH treatments, the MFH effects on cells, however, largely decreased through heat spreading to the culture medium. On controlling and decreasing the volume of culture medium, the problem of heat spreading was solved. It can be consequently expected that PSS-MNPs would be a prospective agent for intracellular cancer magnetotherapy.

Utilization of a Gas-Sensing System to Discriminate Smell and to Monitor Fermentation during the Manufacture of Oolong Tea Leaves.

The operational duration of shaking tea leaves is a critical factor in the manufacture of oolong tea; this duration influences the formation of its flavor and fragrance. The current method to control the duration of fermentation relies on the olfactory sense of tea masters; they monitor the entire process through their olfactory sense, and their experience decides the duration of shaking and setting. Because of this human factor and olfactory fatigue, it is difficult to define an optimum duration of shaking and setting; an inappropriate duration of shaking and setting deteriorates the quality of the tea. In this study, we used metal-oxide-semiconductor gas sensors to establish an electronic nose (E-nose) system and tested its feasibility. This research was divided into two experiments: distinguishing samples at various stages and an on-line experiment. The samples of tea leaves at various stages exhibited large differences in the level of grassy smell. From the experience of practitioners and from previous research, the samples could be categorized into three groups: before the first shaking (BS1), before the shaking group, and after the shaking group. We input the experimental results into a linear discriminant analysis to decrease the dimensions and to classify the samples into various groups. The results show that the smell can also be categorized into three groups. After distinguishing the samples with large differences, we conducted an on-line experiment in a tea factory and tried to monitor the smell variation during the manufacturing process. The results from the E-nose were similar to those of the sense of practitioners, which means that an E-nose has the possibility to replace the sensory function of practitioners in the future.

Synergic Effect of Novel WS2 Carriers Holding Spherical Cobalt Ferrite @cubic Fe3O4 (WS2/s-CoFe2O4@c-Fe3O4) Nanocomposites in Magnetic Resonance Imaging and Photothermal Therapy for Ocular Treatments and Investigation of Corneal Endothelial Cell Migration.

The design of novel materials to use simultaneously in an ocular system for driven therapeutics and wound healing is still challenging. Here, we produced nanocomposites of tungsten disulfide carriers with spherical cobalt ferrite nanoparticles (NPs) as core inside a cubic iron oxide NPs shell (WS2/s-CoFe2O4@c-Fe3O4). Transmission electron microscopy (TEM) confirmed that 10 nm s-CoFe2O4@c-Fe3O4 NPs were attached on the WS2 sheet surfaces. The cytotoxicity of the WS2 sheets and nanocomposites were evaluated on bovine cornea endothelial cells (BCECs) using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay for a duration of three days. The MTT assay results showed low toxicity of the WS2 sheets on BCECs by 67% cell viability at 100 µg/mL in 24 h, while the nanocomposites show 50% cell viability in the same conditions. The magnetic resonance imaging (MRI) of nanocomposites revealed the excellent T2-weighted imaging with an r2 contrast of 108 mM-1 S-1. The in vitro photothermal therapy based on WS2 sheets and WS2/s-CoFe2O4 @c-Fe3O4 nanocomposites using 808 nm laser showed that the maximum thermal energy dispatched in medium at different applied power densities (1200 mw, 1800, 2200, 2600 mW) was for 0.1 mg/mL of the sample solution. The migration assay of BCECs showed that the wound healing was approximately 20% slower for the cell exposed by nanocomposites compared with the control (no exposed BCECs). We believe that WS2/s-CoFe2O4@c-Fe3O4 nanocomposites have a synergic effect as photothermal therapy agents for eye diseases and could be a target in an ocular system using MRI.

Synthesis and characterization of magnetic nanoparticles coated with polystyrene sulfonic acid for biomedical applications.

The development of novel magnetic nanoparticles (MNPs) with satisfactory biocompatibility for biomedical applications has been the subject of extensive exploration over the past two decades. In this work, we synthesized superparamagnetic iron oxide MNPs coated with polystyrene sulfonic acid (PSS-MNPs) and with a conventional co-precipitation method. The core size and hydrodynamic diameter of the PSS-MNPs were determined as 8-18 nm and 50-200 nm with a transmission electron microscopy and dynamic light scattering, respectively. The saturation magnetization of the particles was measured as 60 emu g-1 with a superconducting quantum-interference-device magnetometer. The PSS content in the PSS-MNPs was 17% of the entire PSS-MNPs according to thermogravimetric analysis. Fourier-transform infrared spectra were recorded to detect the presence of SO3 - groups, which confirmed a successful PSS coating. The structural properties of the PSS-MNPs, including the crystalline lattice, composition and phases, were characterized with an X-ray powder diffractometer and 3D nanometer-scale Raman microspectrometer. MTT assay and Prussian-blue staining showed that, although PSS-MNPs caused no cytotoxicity in both NIH-3T3 mouse fibroblasts and SK-HEP1 human liver-cancer cells up to 1000 µg mL-1, SK-HEP1 cells exhibited significantly greater uptake of PSS-MNPs than NIH-3T3 cells. The low cytotoxicity and high biocompatibility of PSS-MNPs in human cancer cells demonstrated in the present work might have prospective applications for drug delivery.

Frequency Shift of a SH-SAW Biosensor with Glutaraldehyde and 3-Aminopropyltriethoxysilane Functionalized Films for Detection of Epidermal Growth Factor.

The frequency shift of a shear-horizontal surface-acoustic-wave (SH-SAW) biosensor in which the concentration of biomolecule is determined by the amount of its adsorption on the sensing film was studied. Simulation results were compared with experimental results to investigate its sensitivity and to develop a model to estimate the concentration of a cancer-related biomarker antigen epidermal growth factor (EGF) in the sample, with two types of sensing films, 3-aminopropyltriethoxysilane (APTES) and glutaraldehyde. With the concentration of the targeted biomarker varying from 0.2 to 5 ng/mL, a typical exponential relationship was found between the concentration and the frequency shift of the SH-SAW sensor. Measurement results showed a clear response of this immunosensor to the mass-loading effects of the antibody-antigen. The sensitivity of the glutaraldehyde film is greater than that of the APTES film owing to the chemisorption of the antibody. In the simulation, a shift of the SH-SAW resonant frequency due to added mass occurred on applying an incremental surface mass density on the sensing film, while in real applications, the concentration of the targeted biomarker to be absorbed in the sensing film is demanded. An empirical model was proposed to calculate the frequency shift in the simulation of the SH-SAW biosensor, corresponding to the concentration of specific biomolecules absorbed on a specific film. From the semi-empirical model, the sensitivity level is found to be 0.641 and 1.709 kHz/(ng/mL) for APTES and glutaraldehyde sensing films, respectively, at a biomarker concentration of less than 1 ng/mL. The developed method is useful for quickly estimating the frequency shift with respect to the concentration of the target molecules in the simulation for SH-SAW sensors.

Using a Dielectrophoretic Microfluidic Biochip Enhanced Fertilization of Mouse Embryo in Vitro.

Droplet microfluidics has appealed to many interests for its capability to epitomize cells in a microscale environment and it is also a forceful technique for high-throughput single-cell epitomization. A dielectrophoretic microfluidic system imitates the oviduct of mammals with a microchannel to achieve fertilization in vitro (IVF) of an imprinting control-region (ICR) mouse. We applied a microfluidic chip and a positive dielectrophoretic (p-DEP) force to capture and to screen the sperm for the purpose of manipulating the oocyte. The p-DEP responses of the oocyte and sperm were exhibited under applied bias conditions (waveform AC 10 Vpp, 1 MHz) for trapping 1 min. The insemination concentration of sperm nearby the oocyte was increased to enhance the probability of natural fertilization through the p-DEP force trapping. A simulation tool (CFDRC-ACE+) was used to simulate and to analyze the distribution of the electric field. The DEP microfluidic devices were fabricated using poly (dimethylsiloxane) (PDMS) and ITO (indium tin oxide)-glass with electrodes. We discuss the requirement of sperm in a DEP microfluidic chip at varied concentrations to enhance the future rate of fertilization in vitro for an oligozoospermia patient. The result indicates that the rate of fertility in our device is 17.2 ± 7.5% (n = 30) at about 3000 sperms, compatible with traditional droplet-based IVF, which is 14.2 ± 7.5% (n = 28).

Extraction of Cell-free Dna from An Embryo-culture Medium Using Micro-scale Bio-reagents on Ewod.

As scientific and technical knowledge advances, research on biomedical micro-electromechanical systems (bio-MEMS) is also developing towards lab-on-a-chip (LOC) devices. A digital microfluidic (DMF) system specialized for an electrowetting- on-dielectric (EWOD) mechanism is a promising technique for such point-of-care systems. EWOD microfluidic biochemical analytical systems provide applications over a broad range in the lab-on-a-chip field. In this report, we treated extraction of cell-free DNA (cf-DNA) at a small concentration from a mouse embryo culture medium (2.5 days & 3.5 days) with electro-wetting on a dielectric (EWOD) platform using bio-reagents of micro-scale quantity. For such extraction, we modified a conventional method of genomic-DNA (g-DNA) extraction using magnetic beads (MB). To prove that extraction of cf-DNA with EWOD was accomplished, as trials we extracted designed-DNA (obtained from Chang Gung Memorial Hospital (CGMH), Taiwan which shows properties similar to that of cf-DNA). Using that designed DNA, extraction with both conventional and EWOD methods has been performed; the mean percentage of extraction with both methods was calculated for a comparison. From the cycle threshold (Ct) results with a quantitative polymerase chain reaction (q-PCR), the mean extraction percentages were obtained as 14.8 percent according to the conventional method and 23 percent with EWOD. These results show that DNA extraction with EWOD appears promising. The EWOD extraction involved voltage 100 V and frequency 2 kHz. From this analysis, we generated a protocol for an improved extraction percentage on a EWOD chip and performed cf-DNA extraction from an embryo-culture medium (KSOM medium) at 3.5 and 2.5 days. The mean weight obtained for EWOD-extracted cf-DNA is 0.33 fg from the 3.5-day sample and 31.95 fg from the 2.5-day sample. All these results will pave a new path towards a renowned lab-on-a-chip concept.